Anal Bioanal Chem DOI 10.1007/s00216-014-8288-4

RESEARCH PAPER

Determination of sulfonamides in butter samples by ionic liquid magnetic bar liquid-phase microextraction high-performance liquid chromatography Lijie Wu & Ying Song & Mingzhu Hu & Xu Xu & Hanqi Zhang & Aimin Yu & Qiang Ma & Ziming Wang

Received: 23 September 2014 / Revised: 17 October 2014 / Accepted: 20 October 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract A novel, simple, and environmentally friendly pretreatment method, ionic liquid magnetic bar liquid-phase microextraction, was developed for the determination of sulfonamides in butter samples by high-performance liquid chromatography. The ionic liquid magnetic bar was prepared by inserting a stainless steel wire into the hollow of a hollow fiber and immobilizing ionic liquid in the micropores of the hollow fiber. In the extraction process, the ionic liquid magnetic bars were used to stir the mixture of sample and extraction solvent and enrich the sulfonamides in the mixture. After extraction, the analyte-adsorbed ionic liquid magnetic bars were readily isolated with a magnet from the extraction system. It is notable that the present method was environmentally friendly since water and only several microliters of ionic liquid were used in the whole extraction process. Several parameters affecting the extraction efficiency were investigated and optimized, including the type of ionic liquid, sample-to-extraction solvent ratio, the number of ionic liquid magnetic bars, extraction temperature, extraction time, salt concentration, stirring speed, pH of the extraction solvent, and desorption conditions. The recoveries were in the range of 73.25–103.85 % and the relative standard deviations were lower than 6.84 %. The experiment Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-8288-4) contains supplementary material, which is available to authorized users. L. Wu : Y. Song : M. Hu : H. Zhang : A. Yu : Z. Wang (*) College of Chemistry, Jilin University, 2699 Qianjin Street, 130012 Changchun, China e-mail: [email protected] X. Xu Department of Chemistry, Liaoning University, 110036 Shenyang, China Q. Ma Chinese Academy of Inspection and Quarantine, 100123 Beijing, China

results indicated that the present method was effective for the extraction of sulfonamides in high-fat content samples. Keywords Liquid-phase microextraction . Ionic liquid magnetic bar . Sulfonamides . Butter . High-performance liquid chromatography

Introduction Sulfonamides (SAs) are synthetic antibiotics often used to prevent infections, treat diseases, and promote growth [1]. However, such use leads to sulfonamide residues in different edible animal products like meat, milk, egg, and fish [2, 3]. The sulfonamide residues in food are of concern because some of them can promote the development of antibioticresistant bacteria, cause allergic reactions in human, and even possess carcinogenic potency [4, 5]. Considering the public health and food safety, the European Union (EU) has set the maximum residue limit (MRL) at 100 μg/kg of total sulphonamide concentration in edible tissues [6]. Therefore, it is imperative to develop reliable, highly sensitive, and easily operated methods for the determination of SA residues in different kinds of samples. Nowadays, it is well known that milk is a very important food consumed globally, because it is highly nutritious, inexpensive, and readily available for promoting growth in children and general good health of the population. However, the presence of antibiotic residues in milk gives rise to public health concerns [7, 8]. Butter is a kind of dairy product. Over the years, the events of the illegal addition of SAs and the overuse of antibiotics in dairy products raised extensive concern about the safety, quality, and security of dairy products. A variety of analytical methods have been reported for the simultaneous determination of SAs in dairy products [9–13]. From the literature review, we found that most of the

L. Wu et al.

published analytical methods were focused on the monitoring of SA residues in milk, but few researches were focused on SAs in butter. One kilogram of milk may usually produce approximately 3 g of butter with a high-fat content [14]. Therefore, the determination of residual SAs in butter became a challenge because of the complexity of biomatrices and the low concentration in samples. Due to the high-fat content of the sample, the removal of the residual lipid from the sample and the cleanup are required before determination. Liquid– liquid extraction (LLE) [15, 16], solid-phase extraction (SPE) [17, 18], and matrix solid-phase dispersion (MSPD) [19–21] were usually applied. However, there are some disadvantages of these methods, such as a large volume of toxic and expensive solvent, time-consuming operation, and reduced frequency of analysis. Nowadays, several microextraction techniques by reducing organic solvent consumption as well as allowing sample extraction and preconcentration to be performed in a single step have been used, such as solid-phase microextraction (SPME) [22, 23], stir bar sorptive extraction (SBSE) [24, 25], and liquid-phase microextraction (LPME) [26–29]. However, SPME and SBSE are time-consuming and the coated fibers or bars are generally expensive and easily destroyed. LPME is a solvent miniaturized procedure of LLE, which has a high preconcentration ability. Hollow fiber liquid-phase microextraction (HF-LPME) is an LPME-based technique and has more advantages than LPME [30–33]. On one hand, microliters of organic solvent were used in HF-LPME; on the other hand, a hollow fiber has an excellent sample cleanup ability, so that large molecules cannot penetrate through the pore in the hollow fiber. Pedersen-Bjergaardand and Rasmussen first reported the U-shaped HF-LPME [34]. The most common format is the hollow fiber fixed to a microsyringe during extraction so that the final extract may be withdrawn into the syringe and directly analyzed [35–37]. Solvent bar microextraction (SBME) was developed by Jiang and Lee in 2004 [38]. In this procedure, 8 μL of organic solvent was held within a hollow fiber with its two ends carefully sealed. This solvent bar can be directly placed into the sample solution for extraction. Due to the free movement of the solvent bar in the sample solution, rapid extraction equilibrium can be achieved. However, the preparation of solvent bars was complex and time-consuming. Hultgren et al. [39] fixed the fiber on a 4 cm metal rod from its end for keeping it at a fixed position in the stirring sample. After the extraction with the help of a magnetic stirrer, the rod was removed from the sample and tweezers were used to put the fiber into methanol for desorption. Yu et al. proposed dual solvent-stir bars microextraction (DSSBME), in which hollow fibers were fixed in a stainless steel wire and could stir by itself [40]. Xu et al. designed a hollow fiber-based stirring extraction bar, where the pores of the fiber wall were impregnated with the organic solvent. Although the

development of the HF-LPME is rapid, the application of these techniques for high-fat samples is less explored [41]. In the hollow fiber-based LPME, appropriate selection of the organic extraction solvent is of great importance to obtain efficient extraction. It is well known that ionic liquids (ILs) are recognized as a greener alternative to the conventional organic solvents in analytical chemistry, especially in extraction and separation [42, 43]. Compared with the traditional organic solvents, ILs show unique advantages of high boiling point and viscosity, low water solubility, and good thermostability as well as the ability of being directly used for reversed-phase HPLC [44]. Previous studies [45, 46] demonstrated that the ILs could be firmly immobilized in the micropores of a supported membrane. Furthermore, ILs have a high affinity to transport some organic compounds selectively [47, 48]. To our knowledge, the application of ILs for extracting SAs has yet to be investigated successfully. The ILs are the suitable extraction solvent for the separation and preconcentration of SAs. This paper aims to present a new extraction alternative that provides a simple and easy method to extract analytes from the complex matrices. An ionic liquid magnetic bar liquidphase microextraction (IL-MB-LPME) was first applied for the extraction of eight SAs in butter samples. For the reasons above-mentioned, three innovations are sought in this study: firstly, the IL-MB-LPME devices were cheaply manufactured and easily assembled. It was prepared only by inserting a stainless steel wire into a polypropylene hollow fiber and immobilizing IL in the micropores of the hollow fiber. The stainless steel wire was not only used as the magnet stirrer, but also achieved magnetic separation which was isolated from the sample matrix easily by an external magnetic field. This smart device made extraction, cleanup, and pre-enrichment into one step, and the solvent bar does not float in the sample solution. Secondly, the polar and nonvolatile IL 1-octyl-3methylimidazolium hexafluorophosphate ([C8MIM][PF6]) was used as the extraction solvent for IL-MB-LPME. The present method was environmentally friendly, can be easily operated, and reliable. Finally, the present method was successfully applied to the treatment of the butter samples, and no other pre-extraction efforts were required. The effects of various experimental parameters were studied and optimized. A rapid, simple, and effective method for the determination of SAs in butter samples was established.

Experimental Chemicals and reagents The standards of sulfacetamide (STD), sulfamerazine (SMR), sulfameter (SMD), sulfathiazole (SMX), sulfachloropyridazine (SCP), sulfadoxine (SDX),

Determination of sulfonamides in butter samples using IL-MB-LPME

sulfamethoxazole (SMZ), and sulfaphenazole (SPP) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The chemical structures of the compounds are shown in Table 1. The mixed stock solution containing the analytes was prepared monthly by dissolving an appropriate amount of SAs in methanol and stored in amber glass bottle at 4 °C. The mixed working solutions were obtained daily by appropriately diluting the stock solution with methanol. Chromatographic grade methanol was purchased from Fisher Corporation (Pittsburgh, PA, USA). Analytical grade anhydrous sodium sulfate, n-

hexane, acetone, and ethyl acetate were purchased from Beijing Chemical Co. (Beijing, China). Pure water was obtained with a Milli-Q water system (Millipore, Billerica, MA, USA). All the solvents and solutions were passed through a 0.45 μm nylon filter (Jinteng Instrument Co., Tianjin, China) before they were used. 1-Butyl-3-methylimidazolium hexafluorophosphate ([C 4 MIM][PF 6 ]), 1-hexyl-3methylimidazolium hexafluorophosphate ([C6MIM][PF6]), and 1-octyl-3-methylimidazolium hexafluorophosphate ([C8MIM][PF6]) were purchased from Chengjie Chemical Co., Ltd. (Shanghai, China).

Table 1 Chemical structures and some physicochemical properties of SAs

Molecular weight

logKowa

pKa1b

pKa2c

Sulfacetamide (STD) 144-80-9

214.2

-0.96



5.4

Sulfamerazine (SMR) 127-79-7

264.3

0.14

2.71

6.77

Sulfameter (SMD) 651-06-9

280.3

0.41

1.88

5.90

Sulfathiazole (SMX) 72-14-0

255.3

0.05

1.85

5.60

284.7

0.31

1.90

5.40

Sulfadoxine (SDX) 2447-57-6

310.3

0.7

2.0

7.1

Sulfamethoxazole (SMZ) 723-46-6

253.3

0.89

1.83

5.57

314.4

1.52

1.90

6.50

Analytes name CAS no.

Sulfachloropyrida -zine (SCP) 80-32-0

Sulfaphenazole (SPP) 526-08-9

a

Structure

logKow values are from the literature [64, 65]

b

pKa1 is the dissociation constant of the amino group which is the basic dissociation constant and the values are from the literature [66, 67]

c

pKa2 is the dissociation constant of the sulfanilamido group which is the acidic dissociation constant and the values are from the literature [66, 67]

L. Wu et al.

Q 3/2 Accurel polypropylene hollow fiber membrane (∼66 % porosity) was obtained from Membrana GmbH (Wuppertal, Germany). The inner diameter was 600 μm, the thickness of the wall was 200 μm, and the pore size was 0.2 μm. The stainless steel wire (505 μm outer diameter) just fitted the hollow fiber membrane.

Instruments Chromatographic separation and determination of the SAs were carried out on the 1100 series liquid chromatograph (Agilent Technologies Inc., USA) equipped with the quaternary gradient pump. An eclipse SB-C18 column (3.5 μm, 4.6 mm×50 mm, Agilent, USA) was used.

Sample preparation The butter samples were purchased from a local large-scale supermarket and stored at −20 °C in a refrigerator before analysis. In the study, five kinds of butter samples produced in New Zealand (sample 1), France (sample 2), China (sample 3), Argentina (sample 4), and Denmark (sample 5), respectively, were used. To prepare the spiked sample, 30 g of sample was weighed and melted at 40 °C in a water bath, and then 1.5 mL of standard working solution was added into the melted sample. The mixture was homogenized by stirring for 30 min and then letting it stand for 1 h at room temperature in the dark. Except for the experiments mentioned in the “Analysis of real sample” section, which were performed with all five samples, all other experiments were performed with sample 1.

IL-MB-LPME procedure The extraction procedure of SAs from the butter sample was depicted in Fig. 1B. First, the eight IL-MBs were simultaneously immersed in 6 mL of 3 mol/L Na2SO4 aqueous solution (extraction solvent) in a 20 mL vessel. Then the butter sample was put into the vessel. The vessel was carefully closed and put into a water bath on the magnetic stirrer. The extraction was performed at 45 °C, and the IL-MBs were continuously stirred at a stirring speed of 500 rpm. After 25 min, with the help of an external magnet, the IL-MBs were separated rapidly from the sample solution. Then the bars were washed with 1 mL of hexane, and the analytes were eluted with 200 μL of methanol in an ultrasonic bath for 3 min. The resulting methanol solution was separated from the IL-MBs with a magnet. After dehydration with anhydrous sodium sulfate (100 mg), the supernatant was rapidly passed through a 0.22 μm PTFE filter membrane, and 20 μL of the resulting analytical solution was injected into the HPLC for analysis. Chromatographic conditions Separation of the analytes was performed using gradient elution at a flow rate of 0.5 mL/min at 35 °C of column temperature. Mobile phases A and B were acetonitrile and 0.5 % acetic acid. The gradient condition is as follows: 0–5 min, 15– 25 % A; 5–12 min, 25–30 % A; 12–22 min, 30–35 % A; 22– 28 min, 35–15 % A; and 28–30 min, 15 % A. The monitoring wavelength was set at 270 nm, and the injection volume of the analytical solution was 20 μL.

Results and discussion IL-MB-LPME device The IL-MB consists of the hollow fiber and stainless steel wire (Fig. 1a). The hollow fiber was cut into segments of 1.2 cm length with a porous volume of ∼4.0 μL (porosity of ∼66 %) [49], and the stainless steel wire was cut into the same length. Before extraction, the hollow fiber and the stainless steel wire were ultrasonically cleaned in acetone for 10 min to remove any possible contaminants and then dried in the air. In order to prepare the extraction bar, the stainless steel wire was inserted into the middle of the hollow fiber. The resulting fiber piece was dipped into the IL ([C8MIM][PF6]) for 30 min for the IL to impregnate the pores of the fiber wall. Since the hollow fiber was hydrophobic, the fiber pores could be filled with IL. In order to remove the extra IL from the surface of the fiber, the fiber was rinsed with water. Each IL-MB was prepared immediately before use and used for only one time to reduce the memory effect.

In traditional magnetic solid-phase extraction, the magnetic adsorbents can be evenly dispersed in the sample solution with the assistance of a disperser solvent and can be quickly separated from the bulk of the sample with a magnet after extraction [50]. This process greatly shortens the extraction time and improves the extraction efficiency. However, the preparation of magnetic adsorbents was complex, difficult, and time-consuming. Besides, up to now, magnetic solid-phase extraction has been demonstrated to be suitable for the treatment of liquid samples and not suitable for solid samples, such as cereals, vegetables, oils, meat, etc. In the present method, the butter sample was dispersed in the aqueous solution by stirring with magnetic solvent bars. The aqueous solution was used both as the extraction solvent to extract the target analytes from butter samples and as the sample solution in LPME. The magnetic solvent

Determination of sulfonamides in butter samples using IL-MB-LPME

Fig. 1 Ionic liquid magnetic bar (A) and extraction procedure of SAs from butter sample (B)

bar was used as magnetic adsorbent to extract the analytes from the sample solution. The target analytes were simultaneously extracted and concentrated in one step. Finally, the magnetic solvent bars were easily isolated from the sample matrix. Optimization of IL-MB-LPME operation parameters In order to obtain the most favorable extraction conditions, the IL-MB-LPME parameters that affect the extraction efficiency, including the type of ionic liquid, sample-toextraction solvent volume ratio, the number of IL-MBs, extraction temperature, extraction time, salt concentration, stirring speed, pH of the extraction solvent, and desorption conditions were investigated. All experiments were performed in triplicate and the concentration of SAs in the spiked samples was 50 μg/kg. Effect of IL type The selection of organic solvent is of major importance in LPME in order to obtain efficient extraction. The ideal organic solvent must easily be immobilized on the hollow fiber and be immiscible with the aqueous phase. Based on these considerations and our previous experience, IL was selected for subsequent experiments. In this

study, three kinds of ILs, [C4MIM][PF6], [C6MIM][PF6], and [C8MIM][PF6], were examined. It was necessary to consider the relationship of the extraction efficiency and the length of the alkyl chain of IL [51]. The characteristics of ILs are listed in Table 2. The experimental results are shown in Fig. 2. The highest extraction efficiencies for all analytes are obtained with [C8MIM][PF6], followed with [C6MIM][PF6], and then with [C4MIM][PF6]. The solubilities of [C4MIM][PF6], [C6MIM][PF6], and [C8MIM][PF6] in water are 18.8, 7.5, and 2.0 μg/L. The viscosities of [C4MIM][PF6], [C6MIM][PF6], and [C8MIM][PF6] are 450, 585, and 710 mPa s, respectively. The extraction efficiency is directly related to the solubility of IL in water. The lower the solubility, the higher the extraction efficiency. The transfer is related to the viscosity. The high viscosity is not beneficial to the transfer of the analytes. Although the viscosity of [C8MIM][PF6] is higher than those of [C4MIM][PF6] and [C6MIM][PF6], the solubility of [C8MIM][PF6] is much lower than those of [C4MIM][PF6] and [C6MIM][PF6]. On the other hand, because SAs belong to the polar compounds, [C8MIM][PF6] has higher polarity than those of [C4MIM][PF6] and [C6MIM][PF6]. Based on these considerations, [C8MIM][PF6] should be most suitable and was selected as the extraction solvent in the subsequent experiments.

L. Wu et al. Table 2 Physicochemical properties of the investigated ILs

IL

Molecular weight

Density (g/mL)

Viscosity (25 °C, cP)

Solubility in water (μg/L)

[C4MIM][PF6] [C6MIM][PF6] [C8MIM][PF6]

284.18 312.24 340.29

1.36 1.29 1.24

450 585 710

18.8 7.5 2.0

Effect of sample-to-extraction solvent ratio

Effect of extraction temperature

Several sample-to-extraction solvent ratios were tested to investigate the influence on the recoveries of SAs. In those tests, the amount of sample was fixed at 1 g. The results are shown in Electronic Supplementary Material (ESM) Fig. S1. Most of the recoveries of SAs were the highest when the ratio was 1:6 (g/mL). Therefore, 1:6 (g/mL) was considered as the optimal sample-to-extraction solvent ratio for the IL-MB-LPME.

The extraction temperature plays a significant role in LPME and SPME [52, 53]. In order to investigate the effect of temperature on extraction efficiency, a series of experiments was carried out at 25, 35, 45, 55, and 65 °C. The effect of temperature on extraction efficiency is shown ESM Fig. S2. It can be seen that the recoveries of SAs are the highest when the extraction temperature is held at 45 °C, except for those of STD and SMX. The increase of temperature can improve the diffusion rate of the analytes and shorten the equilibrium time [54, 55]. However, the increase of temperature can result in the increase of solubility of ILs in the sample solution, which results in the decrease of extraction efficiency. Based on the experimental results, the temperature selected was 45 °C.

Effect of the number of IL-MBs The volume of IL depends on the number of IL-MBs. In this study, the effect of the number of IL-MBs was evaluated and the results are shown in Fig. 3. It can be seen that recoveries of analytes increase obviously with the increase of the number of IL-MBs from 1 to 8 and then do not obviously change with the further increase of the number of bar. The reason may be that eight bars were enough to extract the analytes. Therefore, considering the size of the vessel and the volumes of extraction and desorption solvents, eight bars were finally selected. Fig. 2 Effect of type of IL on the recoveries of SAs. Sample-toextraction solvent ratio, 1:6 (g/mL); number of IL-MB, 8; extraction temperature, 45 °C; extraction time, 25 min; salt concentration, 3 mol/L Na2SO4; stirring speed, 500 rpm; pH of the extraction solvent, 4.5; desorption solvent, methanol; desorption time, 3 min

Effect of extraction time The effect of the extraction time on the extraction efficiency was studied from 15 to 35 min, and the experimental results are shown in Fig. 4. It can be seen that the recoveries increase obviously with the increase of extraction time. The recoveries of most analytes increase significantly when the extraction

Determination of sulfonamides in butter samples using IL-MB-LPME Fig. 3 Effect of the number of MSB on the recoveries of SAs. Type of IL, [C8MIM][PF6]; sample-to-extraction solvent ratio, 1:6 (g/mL); extraction temperature, 45 °C; extraction time, 25 min; salt concentration, 3 mol/L Na2SO4; stirring speed, 500 rpm; pH of the extraction solvent, 4.5; desorption solvent, methanol; desorption time, 3 min

time increases from 15 to 25 min and then reaches a plateau, indicating that the equilibrium has been reached. Above 30 min, the recoveries of most analytes decrease. LPME is not process dependent on exhaustive extraction but an equilibrium partition of the analyte between the extraction solvent and sample solution [38]. When the extraction reached equilibrium, the largest amount of analytes was extracted and the best extraction efficiency and repeatability can be achieved. Generally speaking, a long extraction time is beneficial to the establishment of the extraction equilibrium. However, too long extraction time may conceivably lead to the loss of IL impregnated in the pores of the hollow fiber as was reported in a previous study [46]. Thus, 25 min was selected as the most favorable extraction time. Fig. 4 Effect of extraction time on the recoveries of SAs. Type of IL, [C8MIM][PF6]; sample-toextraction solvent ratio, 1:6 (g/mL); number of IL-MB, 8; extraction temperature, 45 °C; salt concentration, 3 mol/L Na2SO4; stirring speed, 500 rpm; pH of the extraction solvent, 4.5; desorption solvent, methanol; desorption time, 3 min

Effect of salt concentration The salting-out effect has been widely used to enhance the extraction efficiency of polar compounds in extraction and microextraction techniques [56–59]. In this study, the effect of salt concentration on extraction efficiency was examined by adding anhydrous sodium sulfate (Na2SO4) into the extraction solvent in the range of 0.0∼3.0 mol/L. As shown in ESM Fig. S3, the extraction recoveries of all analytes increase in the presence of the salt, and the RSD values of the recoveries decrease from 7.84 to 1.34 %. Therefore, 3 mol/L Na2SO4 aqueous solution (approximately saturated at 45 °C) was chosen as the extraction solvent in the study.

L. Wu et al.

Effect of stirring speed The stirring of the sample solution can shorten extraction time that is required to reach the thermodynamic equilibrium. In this method, the IL-MB could be stirred by itself during the IL-MB-LPME. The effect of stirring speed on the extraction efficiency was investigated. It can be clearly seen from ESM Fig. S4 that the optimal stirring speed is 500 rpm. The high stirring speed can accelerate the mass transfer and result in high extraction efficiency. But too high stirring speed can cause many bubbles to attach to the surface of the hollow fiber and made the butter attach to the surface of the hollow fiber directly, which is not beneficial to the extraction efficiency and precision [38, 60]. Additionally, too high stirring speed may conceivably lead to a loss of IL impregnated in the pores of the hollow fiber. Thus, 500 rpm was considered as the appropriate stirring speed. Effect of pH SAs are ampholytes. A sulfonamide contains one basic amine group (–NH2) and one acidic group (–NH–SO2–) which correspond to pKa1 and pKa2, respectively (Table 1). Typically, high extraction efficiency can be obtained when analytes are in its neutral forms [61]. Theoretically, it is feasible to control the ionizing and nonionizing forms of SAs by adjusting the pH. So the pH of the extraction solvent played an important role in enhancing the extraction efficiency of the target analyte. The results shown in Fig. 5 indicate that the extraction efficiency is highest at pH 4.5, which is in agreement with the results previously Fig. 5 Effect of pH of the extraction solvent on the recoveries of SAs. Type of IL, [C8MIM][PF6]; sample-toextraction solvent ratio, 1:6 (g/mL); number of IL-MB, 8; extraction temperature, 45 °C; extraction time, 25 min; salt concentration, 3 mol/L Na2SO4; stirring speed, 500 rpm; desorption solvent, methanol; desorption time, 3 min

reported by Tao et al. [62] and Guo et al. [59]. When pH is adjusted to the average value of pKa1 and pKa2 of SAs, the neutral molecule is the dominant species [63]. In view of the properties of the analytes (Table 1), pKa1 and pKa2 of analytes are in the range of 1.57 to 2.77 and 5.57 to 7.10, respectively; 4.5 was around the average value of pKa1 and pKa2 of the sulfonamides. Hence, a pH 4.5 extraction solvent was selected in the following experiments. Desorption conditions After the extraction was completed, 200 μL of organic solvents including n-hexane, acetone, methanol, and ethyl acetate were used to elute the analytes from the IL-MB. The results showed that n-hexane cannot elute any kind of target analytes, n-hexane had poor desorption performance, and methanol afforded high efficiency for most of the SAs. Therefore, methanol was used as the desorption solvent throughout the experiments. The effect of desorption time was also investigated. The analytes on the magnetic solvent bar were ultrasonically desorbed. The result indicated that 3 min was sufficient for desorption. When the time was too long, the analytes would be lost. So, 3 min was chosen to be the feasible desorption time. Method validation Limits of detection and quantification To evaluate the performances of the present IL-MBLPME, the linearity, limits of detection (LODs), and

Determination of sulfonamides in butter samples using IL-MB-LPME Table 3 Analytical performance Analyte

Regression equationa A=(a±SDa)+(b±SDb) c

Linear range (μg/kg)

Correlation coefficient

LOD (μg/kg)

LOQ (μg/kg)

STD SMR SMD SMX SCP SDX SMZ SPP

A=(0.16±0.23)+(0.25±0.001) c A=(0.71±0.28)+(0.32±0.002) c A=(1.07±0.61)+(0.41±0.004) c A=(0.90±0.30)+(0.42±0.002) c A=(1.49±0.55)+(0.29±0.003) c A=(2.47±0.62)+(0.51±0.004) c A=(1.44±0.42)+(0.59±0.003) c A=(0.95±0.84)+(0.45±0.005) c

6.00∼300.00 6.00∼300.00 6.00∼300.00 6.00∼300.00 6.00∼300.00 6.00∼300.00 6.00∼300.00 6.00∼300.00

0.9985 0.9991 0.9993 0.9987 0.9995 0.9986 0.9996 0.9998

2.17 1.75 1.48 1.87 1.36 1.40 1.30 1.20

7.25 5.86 4.94 6.25 4.53 4.67 4.32 4.00

a

A, peak area of the analyte; c, concentration of the analyte in micrograms per kilogram; a, intercept; b, slope; SDa and SDb, standard deviations of the intercept and slope, respectively

Good linearity was obtained for the eight SAs with the correlation coefficient (r2) ≥0.9985. The LODs (S/N=3) and LOQs (S/N=10) are in the range of 1.20–2.17 and 4.00–7.25 μg/kg, respectively. It can be concluded that the present IL-MB-LPME is a feasible method in extracting SAs from butter samples.

limits of quantitation (LOQs) were tested using the spiked butter samples under the optimum experimental conditions. The working curves were constructed by plotting the peak areas measured versus the concentrations of the analytes in the spiked samples. The linear regression equations and correlation coefficients are listed in Table 3.

Table 4 Analytical results of real samples (n=5) Analyte Spiked concentration (μg/kg)

Sample 1 Recovery (%)

STD

SMR

SMD

SMX

SCP

SDX

SMZ

SPP

Sample 2 RSD (%)

Recovery (%)

Sample 3 RSD (%)

Recovery (%)

Sample 4 RSD (%)

Recovery (%)

Sample 5 RSD (%)

Recovery (%)

RSD (%)

10.00 60.00 100.00 10.00 60.00 100.00 10.00 60.00 100.00

78.4 76.9 79.4 90.4 92.1 93.4 94.9 92.1 95.8

5.3 3.5 3.8 3.6 3.1 2.7 3.8 4.1 3.2

78.3 76.9 74.6 87.6 88.4 90.1 96.4 94.3 93.8

6.8 3.4 3.9 3.2 4.1 3.8 2.3 4.3 3.6

74.2 77.3 74.3 89.2 85.4 84.6 95.3 97.2 90.8

5.1 4.0 5.1 3.6 2.9 3.9 3.4 3.5 2.4

75.1 72.9 74.9 88.2 86.8 85.4 94.8 92.4 95.3

4.8 5.1 3.9 4.2 3.8 3.1 2.4 3.4 3.8

73.2 75.6 74.8 92.4 86.9 90.1 94.4 91.2 93.5

5.0 6.4 6.0 3.4 4.1 3.1 3.0 3.4 3.0

10.00 60.00 100.00 10.00 60.00 100.00 10.00 60.00 100.00 10.00 60.00 100.00 10.00 60.00 100.00

86.4 84.1 86.9 87.1 89.4 90.9 97.1 98.6 101.3 99.1 98.4 102.0 99.7 99.8 101.8

5.4 4.5 4.3 4.3 2.9 2.4 2.4 1.5 2.1 1.3 2.9 3.4 2.6 1.2 3.0

88.2 85.4 87.1 85.7 89.7 90.4 99.4 97.5 101.5 98.4 102.6 95.8 101.2 98.7 98.9

5.4 6.1 5.4 3.4 4.2 3.8 3.9 3.6 3.5 2.1 2.1 2.4 4.3 2.9 3.4

84.5 88.3 85.7 83.5 86.1 84.5 97.6 98.4 96.7 100.3 102.3 99.7 98.1 99.7 101.8

4.2 5.0 4.5 3.8 2.6 3.1 3.4 2.7 2.8 3.9 3.5 4.0 2.4 1.9 2.1

86.8 84.1 82.8 87.1 89.4 88.7 98.1 101.3 97.4 99.7 96.8 103.5 102.3 98.8 100.4

5.0 4.0 5.4 2.6 3.1 3.8 3.4 3.0 2.1 3.0 2.6 2.3 2.4 3.0 2.8

83.7 82.4 79.2 85.4 89.7 87.2 99.4 96.8 94.1 103.5 98.7 101.2 101.8 99.4 96.1

4.9 3.6 3.4 2.1 4.1 3.8 3.1 4.0 2.1 3.5 3.4 2.8 2.4 2.9 3.8

L. Wu et al. Fig. 6 Chromatograms of blank butter sample 1 (a) and spiked sample 1 (b): spiked concentration, 50 μg/kg. 1, STD; 2, SMR; 3, SMD; 4, SMX; 5, SCP; 6, SDX; 7, SMZ; and 8, SPP

Precision and accuracy To evaluate the precision and accuracy of the present method, the samples at a spiked concentration of 50 μg/kg were analyzed. The intraday precision was measured by analyzing a sample five times in 1 day. The interday precision was obtained by analyzing a sample once a day over five consecutive days. The analytical results are shown in ESM Table S1. The recoveries and relative standard deviations (RSDs) are in the range of 76.15–101.46 and 1.39–6.04 %, respectively. The precision and accuracy for the present method should be satisfactory.

Analysis of the real sample Five butter samples were analyzed to test the applicability of the proposed extraction procedure. The analytical results are shown in Table 4. As can be seen, the present method provides good recoveries (73.25–103.85 %) and acceptable precision (

Determination of sulfonamides in butter samples by ionic liquid magnetic bar liquid-phase microextraction high-performance liquid chromatography.

A novel, simple, and environmentally friendly pretreatment method, ionic liquid magnetic bar liquid-phase microextraction, was developed for the deter...
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